Ida S. Owens, PhD, Head, Section on Genetic Disorders of Drug Metabolism

Nikhil K. Basu, PhD, Research Fellow
Bhabadeb Chowdhury, PhD, Research Fellow
Partha Mitra, PhD, Visiting Fellow
Amanda Garza, BS, Postbaccalaureate Fellow

Mammalian UDP-glucuronosyltransferase (UGT) isozymes are critical catalysts for detoxifying endogenous metabolites and numerous potentially injurious lipid-soluble phenols derived from the diet and environment. The isozymes detoxify by conjugating glucuronic acid with metabolites, drugs, toxins, and environmental chemicals to make them water-soluble and excretable. Glucuronidation reactions prevent accumulation of neurotoxic levels of plasma bilirubin, inactivate many drugs, and avert the mutagenicity and carcinogenicity of aromatic hydrocarbons such as benzo(a)pyrene found in cigarette smoke and automobile emissions. Moreover, UGTs prevent accumulation of dietary phenols that inhibit enzymes. On the other hand, extensive glucuronidation can be disadvantageous. Thus, the premature clearance, associated with UGT metabolism, of many orally administered therapeutic drugs is a long standing problem. Metabolism is overcome by administering compensatory higher doses, which can lead to serious side effects, and, for decades, drug inefficiency has been the impetus to developing inhibitor(s) of UGT. The enzymatic mechanism(s) and properties that enable UGTs to convert numerous unrelated lipid-soluble phenols to innocuous glucuronides remain unknown, along with methods to control conjugation and prevent premature clearance of therapeutic drugs. An important research aim, therefore, is to understand the properties and mechanism(s) of this system of detoxification.

Phosphorylation of UGT proteins with signaling

Basu, Mitra, Kole,a Kubotab

While analyzing UGTs to understand glucuronidation requirements in human colon cell lines, we discovered that the isozymes require phosphorylation that is regulated via signaling. Given that phosphorylation is mediated by certain PKC isozymes or tyrosine kinase(s), the action of classical PKC agonists and antagonists as well as the effects of PKC translocation-specific inhibitor peptides confirmed the involvement of signaling events. Furthermore, immunocomplexing of UGT from solubilized human LS180 colon cells with antiUGT traps PKC isozymes. Parallel hydrogen-peroxide (H2O2) stimulation of UGT phosphorylation and glucuronidation activity and inhibition of H2O2-enhanced activity by catalase and herbimycin demonstrate that cellular oxidants are signal(s) for the PKC-UGT system. Specifically, our results demonstrate that UGT1A1, 1A7, and 1A10 undergo phosphorylation by PKC(s), which is, in turn, phosphorylated and activated by tyrosine kinase(s). For UGT2B family members that generally metabolize endogenous substrates, we found evidence of direct phosphorylation by tyrosine kinase. In addition to three PKC phosphorylation sites, we showed that UGT2B7 has two tyrosine phosphorylation sites. Alteration of one or all PKC sites in UGT2B7 that generated a triple-mutant had no effect on activity while a single or double mutation at tyrosine sites caused dominant negative activity. The highly specific PKC inhibitor calphostin-C and general kinase inhibitor curcumin inhibited all UGT activities in cell culture. Given that both herbimycin-C and genistein are tyrosine kinase inhibitors, UGT1A1, 1A7, and 1A10 were sensitive to the former, and UGT2B7 was more sensitive to the latter. In summary, our results demonstrate that UGTs undergo phosphorylation and that UGT1A family members are phosphorylated via PKC, with signaling mediated via oxidative stress. At least one UGT2B family member requires tyrosine phosphorylation.
 

Overexpression of PKCs in conjunction with mutants of UGT1A7 suggested that PKC isozymes phosphorylate specific sites. In addition, evidence derived by comparing normal isozyme-specific peptides with mutant peptides demonstrated that at least three different PKC isozymes can phosphorylate UGT1A7 and 1A10 at positions 73, 202, and 432. Mutations at positions 75 and 112 in UGT1A1 and at positions 73 and 202 in 1A7 and 1A10 were dominant negative while alteration of a third common position, 435 or 432, indicated that the phospho-group(s) plays a unique and novel role in catalysis by operationally controlling pH optima and substrate selection. Alteration of serine-432 to glycine in UGT1A7 caused a dramatic shift in its pH optimum from 8.5 to 6.4 in parallel with the acquisition of new substrate activities or loss of activities resembling substrate selections by 1A10. Substitution of serine-432 in 1A7 with a number of other amino acid residues retained the pH 8.5 optimum seen in the wild-type enzyme. In parallel with in vitro activity, cultures of COS-1 cells expressing S432G-UGT1A7, but not wild-type 1A7, avidly glucuronidated the new substrate 17beta-estradiol, similar to wild-type 1A10 and its S432G mutant. For the first time, we presented evidence that phospho-groups play an operational role in catalysis rather than just causing the typical conformational change in protein structure.

Basu NK, Kole L, Owens IS. Evidence for phosphorylation requirement for human bilirubin UDP-

glucuronosyltransferase (UGT1A1) activity. Biochem Biophys Res Commun 2003;303:98-104.
 

Location and properties of UGT determine control of gastrointestinal chemical absorption

Basu, Kole;a in collaboration with McDonagh  
Using Northern blot, in situ, and biochemical analyses, we determined distribution and function of the isozymes UGT1A1, the bilirubin-metabolizing isozyme, and UGT1A7, 1A8, 1A9, and 1A10 and found them to be segmentally distributed throughout the GI in mucosal epithelia. Recombinant isozymes exhibited a pH optimum of 6.4 or pH 7.6 and/or broad ranges; activity was either unaffected or progressively inhibited by increasing substrate concentrations. All exhibited wide substrate selections for dietary and environmental chemicals but with differing optimal conditions. We demonstrated the impact of glucuronidation on drug and chemical absorption at the GI level by exploiting our recent finding that UGTs require phosphorylation. [33P]Orthophosphate incorporation into immunoprecipitable UGTs confirmed phosphorylation. The simultaneous suppression of UGT radiolabeling and activity in cell lines by the general kinase inhibitor curcumin and the PKC-specific inhibitor calphostin-C also confirmed the requirement for phosphorylation. Using curcumin-treated mouse duodenal loops, we demonstrated that free bilirubin (a marker) uptake was dramatically increased concurrently with decreases in bilirubin-glucuronides in lumen, tissue, and portal blood. The results represent the first direct evidence that UGTs control GI absorption of polyphenols and that maintenance of their phosphorylation is required to limit chemical absorption.
 

Basu NK, Ciotti M, Huang MS, Kole L, Mitra PS, Cho JW, Owens IS. Differential and special properties

of the major human UGT1-encoded gastrointestinal UDP-glucuronosyltransferases enhance potential to control chemical uptake. J Biol Chem 2003; October 13 [Epub ahead of print].

Gong Q-H, Cho JW, Potter C, Gholami N, Huang TH, Basu N, Pennington M, Owens IS. Thirteen

UDP-glucuronosyltransferase genes are encoded at the human UGT1 gene complex locus. Pharmacogenetics 2001;11:357-368.

Sugatani J, Kojima H, Ueda A, Kakizaki S, Yoshinari K, Gong Q-H, Owens IS, Negishi M, Sueyoshi

T. Phenobarbital response enchancer module in UGT1A1, bilirubin UDP-glucuronosyltransferase gene. Hepatology 2001;33:1232-1238.

Transient inhibition of gastrointestinal UGT activity markedly improves mycophenolic acid immunosuppressant activity

Basu, Kole,a Chowdhury

While the promising immunosuppressant mycophenolic acid (MPA) is being widely prescribed for both adult and child renal transplant patients and for autoimmune diseases, serious side effects are associated with high dosage requirements because of extensive glucuronidation of the drug. We found that the cellular distribution and biochemical properties of the primary metabolizers of MPA UGT1A7, 1A8, 1A9, and 1A10 contribute significantly to high oral dose requirements. In situ hybridization studies revealed that UGT1A7-, 1A9-, and 1A10-mRNAs are located in GI mucosal epithelia; studies with microsomes isolated from adjacent specimens showed that esophagus, ileum, duodenum, and colon have moderately high to high glucuronidating activities. Recombinant UGTs avidly glucuronidate MPA, showing nearly linear increases in activity with concentration (up to 800 microM); only UGT1A9 showed typical saturation kinetics. Each isozyme generates about 80 percent ether-linked and about 20 percent acylglucuronide. To establish the in vivo impact of MPA glucuronidation, we used the general kinase inhibitor curcumin and highly specific protein kinase C inhibitor calphostin-C on LS180 colon cells to inhibit UGT's newly discovered phosphorylation requirement. Transient inhibition of glucuronidation by oral pretreatment with curcumin before MPA administration caused a six-fold greater immunosuppression of antigen-stimulated spleen cytotoxic T-lymphocyte proliferation in mice. Hence, glucuronidation can adversely impact drug efficacy. Moreover, curcumin pretreatment to inhibit GI-distributed UGTs represents a model for increasing bioavailability of highly glucuronidated drugs.

COLLABORATORS
Antony McDonagh, PhD, University of California, San Francisco CA
Masahiko Negishi, PhD, Laboratory of Reproductive and Developmental Toxicology, NIEHS, Research Triangle Park NC
aLabanyamoy Kole, PhD, former Postdoctoral Fellow
bShigeki Kubota, MD, former Postdoctoral Fellow

For further information, contact owens@helix.nih.gov